Production of fat-soluble vitamin ester concentrates



preteens- 23, 194

raonuc'rIoN or FAT-SOLUBLE VITAMIN ss'raa concanraa'ras 'ci iarles E.Dryden, East range, and Loren 0;

Boston, Newark, N. 1., assignors to National Oil Products Company.Harrison, .N. 1., a corporaticn'ot New Jersey No Application September28, 19:44, I

' Serial No. 556,284

l I ocialmsfiwtisr-ail The present'invention relates to the produc-'tion of fat soluble vitamin concentrates from natural oils and fatscontaining the same. More partlcularly the present invention relates tothe production of relatively high potency vitamin A concentrates whereinthe vitamin'A is largely in natural ester form from materials of marineorigin containing vitamins A and D.

Various methods of concentrating the 'vitamin content of oils of marineorigin containing vitamins are known in the art, amongthem, processeswhich inc ude saponiflcatio'n or the fatty acid glycerides by means ofcaustic soda or caustic potash. In general, prior art saponificatlonprocesses contemplate complete saponiiication of all the saponiflableconstituents otthe oil in an effort to produce concentrates of vitaminsof the highest potency. In aprocess of this character,- the vitamin andother'higher alcohol esters areconverted to soaps; vitamin alcohols andother higher alcohols. Vitamin alcohols are separated from the residualsoaps and other components of e concentrate thus produced is of a theoils; relatively high potency, but the vitamin is substantiallycompletely in an alcohol form rather than in the natural ester form.Among th disadvantages of such a, process is that the vitamin alcohol ismuch more subject to oxidation and upon storage for any length of timeexhibits a lesser biological potency than the natural ester.

In 0. S. application Serial No.'450,'757, a-process involving a partialsaponification of fish liver oils and the like to produce vitaminconcentrates wherein a substantialportlon oi the original vitamincontent is obtained in ester form is disclosed. The process set forth inthe aforementioned application includes the addition of sufficientalkali to' partially saponify the saponifiable matter in the oil.Thereafter the soap formed is separated as by extracting thenonsaponifled residue with ethylene dichloride and the resultantnonsaponi lied residue after removal of the ethylene dichloride is thenfurther extracted with a polar solvent such as methanol to remove thevitamin A alcohol. The resultant ester-concentrate produced is ofconsiderably higher potency than the original oil. However, there arelimits to the strength of the'ester concentrate produced from anyparticular oil since a higher degreeof saponiflcation tends to hydrolyzea greater amount of the vitamin ester to the vitamin A alcohol. In;racticing the process '0! the aforementioned'application; therefore, thevitamin-containing oil;

is saponified to an extent which will produce-the highest potency esterconcentrate and still hydrolyze only aminor proportion 01 the vitamin Aester'in the original oil to the vitamin alcoq hol. By proceeding inthis manner, it is there-.- iore possible to produce ester concentratesof 5 considerably greater potency than the original 011 while at thesame time hydrolyzing only a small amount of the ester of the originaloil.

Although it has been proposed in accordance with the aforementionedapplication to -reduce the speed of reaction as by limiting the amountof catalyst or the concentration of the alkali and" although the slowerreaction thus eflected has increased the potency oi the vitamin A esterproduced, ithas not been possiblewhen proceedin with the reaction underadiabatic conditions, as

set forth in the prior application, to slow down the speed or reactionsufllciently to greatly increase the potency of the resultant vitaminester concentrates by such means. Inasmuch as the reaction with thealkali is exothermic, the. tem-' perature of the mixture of alkali andthe oil being treated normally rose to a temperature of approximately50-60 during the reaction,'and this was true no matter'how slowly thecaustic was added during saponiflcation. In accordance with v theteachings of the prior art, this was not undessirable, since some of theprior patents even taught the desirability of heating the mixture ofcaustic and oil in order-to produce a more-complete .sa-

poniflcation.

In accordance with the present invention, however, it has now been disvered that ii the reaction mixture-o1 causti and oil is cooled duringthe reaction so that it, is maintained under isothermal conditions andpreferably at temperatures below approximately 35 C., a much higherpotency vitamin ester concentrate is produced and atthe .same time alesser amount of vitamin A ester is hydrolyzed to vitamin A alcohol.

The exact reason for this phenomenon is not clearly understood, but itis evident that that portion of the fatty acid glycerides'whichis notsaponiiled during the incomplete saponiflcation is rendered soluble in apolar solventsuch as methanol, probably due to mono anddiglycerideformation. Thus when the residue after separation of the'soap istreatedwith a polar solvent such as methanol, a relatively largeproport'ion ofthe nonsaponifled nonvitamin content is removed to leave behind a morepotent vitamin ester concentrate. Further, during the reaction a muchlesser quantity of the vitamin ester is hydrolyzed to vitamin alcohol.It has therefore become possible to not only separate the vitamin esterin I relatively large yields from the remaining components of the oil,but ester by means of a simple process so that vitamin esterconcentrates of relatively high potency are produced. Further, higheryields are obtained considering, both vitamin esterv and alcohol ascompared to the total amount of vitamin in the original oil.

'. It is one of the objects of the present invention,

, therefore, to prepare vitamin concentrates from natural vitamincontaining materialsby partial saponification, while cooling the oil soas to proalso to concentrate the duce vitamin ester concentrates ofrelatively high potency.

A second object of thepresent invention fisto provide an'isothermicprocess for the partial saponification of fish liver oils andthe'like,-,whereby a much lesser proportion of. vitamin ester is hydrolyzed to vitamin alcohol.

A third object of the present invention is partially saponify a vitaminester containing marine' material while cooling the same so as to formas large a, proportion as possible of polar solvent soluble materialfrom the unsaponified non-. vitamin components so that upon treatmentwith a polar solvent such as methanol a vitamin ester concentrate of arelatively high potency'remains.

Other objects and advantages cf the present invention will be apparentfrom the present description' and claims.

In general, it is preferable when practicing the present process to addthe caustic at a relatively slowrate. Thus aportion'of the caustic maybe added in relatively concentrated form over a substantial period, andthe additional caustic added in relatively dilute form over anotherperiod;

or, in the alternative, a relatively more dilute caustic may be slowlyadded. In general, the lower the temperature ofreaction, the less thequantityof ester converted to alcohol form, and

the temperature of reaction should be as low as possible depending onthe temperature at which the oil tends to solidify and the reactiontends to "slow up to such an extent that a complete'reaction will nottake place. In general, temperaturesbelow about 0. are used andtemperaturesfrom 0 C. to 25 C. have been found to be particularlysuitable. As may be understood, the

temperature of the reacting mass may be kept at the desired range duringsaponification byany suitable cooling means. f I

The'preseht process is applicableto the production -of vitaminconcentrates from various types of vitamin containing materials andparticularly materials of marine origin containing vitamins A and D.Such oils are the fish liver oils 2 to 5% of isopropanol or other polarcatalyst is'.

' temperatures of the present process. .The speed such. as cod liveroil, ling cod liver oil, pollack liver oil, tuna liver oil, shark liveroil, dogfish liver oil, mackerel liver oil, sword fish liver oil,

spear "fish liver' oil, sole liver oil and halibut liver oil. Marineoils such as sardine oil, pilchard'oil,

herring oil, seal oil and whale oil are also suitable.

In place of the oils which havebeen obtained from the 'raw material bycold pressing processes or other conventional means, solvent extractsand partial concentrates of oils of the character described may also beused as, for example, the isopropanol extracts of the liver andmarine'oils specified.

, tracted from the partially saponifled mass bythe The partialselective'saponification is carried out by treating a vitamin-containingmaterial of the character set forth with caustic in the manner wellknown in the art for preparing vitaminj concentrates, except that theamount 'of caustic usedisa lways insuflicient' to completely saponifythe oil." "Aspointed out in the aforementioned min alcohols is producedthan when proceeding in accordance with the process of the priorapplication. A certain amount of vitamin alcohols, how-' ever, naturallyoccurs-in the oil, and the unsaponified mass contains this smallproportion of vitamin alcohol, a part of the unsaturated glyceridespresent in the original oil un'saponifiable matter, glycerine, soaps,etc. I The present process can be practiced, at least to some degree,with any degree of saponification of the oil up to approximately 98%saponification. In general, the amount of the caustic em'ployed will bethat equivalent to somewhat less; than 98% with the larger amounts ofcaustic increasing the potency of the vitamin alcohol portion extractedby means 'of' methanol and correspondingly tending to hydrolyze agreater p'roportiornofthe vitamin ester to the alcohol. In general,'suiiicient caustic will be employed in the presentproc ess equivalentto the saponiflcation of from 60 to about of the saponifiabie matter inthe oil. The saponification reaction may be retarded by other means inaddition to the temperature control, for example, the oil may bedilutedwith a suitable inert solvent such as a nonpolar solvent on the order ofethylene dichloride, heptane, triohlorethylene, hexane, cyclohexarie,methyl cyclohexane, or a mixture of inert solvents. If a solvent isemployed-the weight of the solvent should range from about 15 to 99% andpreferably from about 25-75% to obtain the most eflicient selectivesaponiflcation.

tarded by reducing the amount of catalyst ordi- Saponiflcation may alsobe renarily employed in the saponification reaction. Usually, insaponifications of this type, fromabout employed. .This allows thesaponiflcat'ion reaction to take place without high heating. v How-vever, from to l% of isopropanol or other cata-' lyst has been foundsatisfactory, even at the low of the reaction may also be retarded byutilizing "less concentrated caustic.

It will be understood from the foregoing that I the present reaction maybe retarded in order to produce the greatest potency ester concentrates'by'the use of a solvent'or diluent of an inert character, by use of alower catalyst concentrat-ion and a lower caustic concentration. Ingeneral a caustic concentration equivalent to 30% to 50% of caustic sodaor potash is suitable. All of these factors, however, do not give riseto the desirable results of the present invention, unlss the.temperature is' also maintained at a relatively low level, as beforestated, below'approximately 35 C.

After the saponification is completef i. in

from 1 to 12' hours, the semi-concentrate is exuse or a nonpolarsolvent. The particular method forthe extraction is described in detailin copending application Serial vNo. 533,256, wherein it is pointed outthat the separation of the soap from the solvent should preferably takeplace at an elevated temperature as, for example, from 40-80 andpreferably from 60-70" C. Further, during this separation which takesplace rapidly at this elevated temperature, the amount. or-

water present should be regulated from 12-35% and, in some instances, ashigh as 50% as compared to the weight of the soap produced. Preferably,the amount oiwater should be determined by the solubility of th soap inthe extraction solvent, preferably ethylene dichloride;

the amount or water being regulated so that from /g to 5% of the soapand preferably somewhat less than 1% is soluble in the ethylenedichloride.

The present low temperature saponification gives rise to vitamin esterconcentrates of the highest plication it is pointed out that-it isdesirable for the extracted vitamin semi-concentrate to remain incontact with alkali for the shortest period of time so that thebiological potency of the "concentrates produced may be enhanced, eventhough in the present process the low temperature saponification takes aconsiderable length of time as compared to relatively hightemperature'saponiflcation, this does not have a deleterious effeet onthe concentrates produced. This is probably due to the low temperatureinvolved. However, after the saponiflcation reaction is complete and themass oi soap and saponiiied matter is heated, as' by the addition oi.hot solvent for fractionation, it is desirable at these'tempera-' turesin the neighborhood of 50-80 C.that the separation of the soap tromthesolvent extract take place as rapidly as possible. The extraction abouthour. The average temperature of theoil and soap mixture during'mixingwas about 13 C. The soap produced was extracted successively withv 500cc. of ethylene dichloride at 65C., a total of six extractions beingmade. The combined extracts were kept at 0 C. overnight, filtered andthe solvent removed under vacuum. The semi-concentrate thus produced hada potency of 331,000 units 01' vitamin A per gram. Thereafter thesemi-concentrate was extracted three times with 5 volumes of methylalcohol, the mixture of alcohol and semi-concentrate being first heatedto 50 C. and then cooled to 18 C.

and decanted. The vitamin content of the residue ester fraction wasequivalent to 58% of the total vitamin A' recovered. The potency of theester fraction was v477,000 unitsof vitamin A per gram.

The total overall vitamih A recovery, i. e. the vitamin A in themethanol extract as well as that in the residue was equal to 97.9% ofthe original vitamin A in the oil. A similar reaction to the above wascarried out, except thatthe oil was not cooled during saponiflcation.The temperature in this case rose. to a maximum of 62 C. in 7 minutes.As compared to the reactionat low temperature, only 38.9% vitamin A wasrecovered in ester form. Although the semi-concentrate produced had apotency of 300,000 units or vitamay then be'carried out a number oftimes with lesser quantities of ethylene dichloride or other suitablesolvent'as' mentioned in the prior appllcation. and the resultantconcentrates combined. Thereafter, the ethylene dichloride is removed byany suitable-means to produce a semiconcentrate. The semi concentratesmay then be fractionated by means of polar solvents or they may befractionated by processes other than selective solvent extraction as,for example, high vacuum distillation, vacuum distillation, absorption,etc. It

is preferred, however. that a fractionation with --polar solvents becarried out and preferably the polar solvent used is methanol orethanol. However, any other solvent which is characterized by beingmiscible with (a solvent for), -vitamin alcohols but immiscible with (anonsolvent for) vitamin esters, may be employed in lieu of or inconjunction with methanol or ethanol.

Such solvents include, among others, isopropanol, n-butanol, acetone anddiacetone alcohol, each of which has been modified by the addition of atleast 9% min A per gram, the potency of the ester fraction i. e'..themethyl extract residue of the semiconcentrate was only 172,000 units ofvitamin A per gram. The overall yield of vitamin Alfrom the high t mprature process was 943%,.

Example II t rams of soupfln" shark liver oil-having 1 103,000-unlts ofvitamin A per gram were placed in a beaker unmersed in ice watermaintained at a'temperature of -3 C. with ice. 0.75%. by

weight of isopropanol was added and then sumcient 49% KQH to saponify90% of the glyc-- erides in the 011, i. c. 33.4 grams of 49% KOH were 1added dropwise while stirring. The KOH was added'during a period of 1hour and the mass was mixed .for an additional /2 hour. Thereafter,sufficient water was added to bring the moisture content of thesoapforined, on a wet' basis, to 23.5%. The average. temperature ofthe'oil during saponiflcation was'3" C. and after a mixing and'additionof water, the mass was stored at 3 C. for 42 hours. Thereafter the-soapI was extracted five times with ethylene dichloride water, methanol,and/or ethanol. As previously pointed out, a large proportion of theunsaponifled material aside from the vitamin esters is reh--deredsoluble in solvents of'the character just described andconsequently the extraction yields a concentrated vitamin ester residue.

E xamplel 100 grams ofa soupfln shark liver "oilcontaining 63,200 unitsof vitamin A per gram. were placed in -a beaker immersed in ice watermain-- tained at a temperature of4 C. with ice. 31.2 grams of 49%KOH,sufficient to saponify 82% of the glycerides in the 'oil, and 15 gramsof water (suiiicient'to make the water contentv of th wet soap produced25% on completion of the reac-' tion) were stirred-in. The reactionmixture was stirred'io'r-2 hours and then allowed to settle for ,at'70"C..'40'0 cc. of. ethylene dichloride being used for each extraction. Thecombined extracts were kept'at 0" C. overnightand then filtered.

The ethylene dichloride was then removed under J a vacuum. Thesemi-concentrate thusproduced was then extracted with methanol in amanner 1 similar to Example I. The following results were obtained:

- Unitsof' vitamin Aper gram Vitamin potency of semi-concentrate-785,000 Vitamin potency of alcohol (methanol extract) 135.060 Vitaminpotency of ester (methanol 9 residue) 868,000

Overall yield of vitamin'A was 9am.

Example 111 100 grams of ling cod oil'having a'potency of 24 4,000unitsof vitamin A per gram were placedj in a-beakerimmersed in icewater'maintained at a temperature of 9 C. with ice. 3% by weight ofisopropanol was added and then sufllcient,

70%: of the-glyoerides in the oil-,i. e. 35.5 grams of-32.7%- KOH. Themass was stirred for a pe- :riod of one hour-and was thereafter storedat 3 C; for -16 hours.-- The average temperature during'.-saponification was 1-G. After storage, the soap was extracted fivetimes with ethylene dichloride render a relatively large proportion ofthe material other than the vitamin ester and soap formed .at 70 C.500cc. of ethylene dichloride were used tor the first extraction and 300cc. for successive extractions.- The combined extracts were kept at 0..C. overnight, filtered and the ethylene dichloride removed under vacuum;The semi-concentrate. thus produced 1 was thenextracted with methanol asin Examples I and II. The followinglresults were obtained:. Units ofvitamin .Aper gram Vitamin potency of -semi-concentrate 605,000 Vitaminpotency .of alcohol. (methanol extract) 256,000 Vitamin potency ofester(methanol residue) 680,000 Overall yield of vitamin-A was 98.6%. v

.EMmpZel-V- 100 grams or halibut liver oil having a potency of92,500units or'vitamin A per gram were placed in aibeaigerimmersed inice water maintained at a temperature of 8 C. with ice. 3% by weight ofisopropanol. was added and then suflicient 36.8% KOH was added whilestirring to saponify 70% of the glycerides in the oil, 1 .e. 32.6% of36.8% KOH. The total time of stirring was ,1

hour, and the average temperature during saponificationof the'oil' 0 C.v Thereafter the mass was stpredfor 18 hours at 3 C. The scan vWasthenextracted five times with ethylene dichloride at 70 C. 500 cc. ofethylene dichloride were used .for the first extraction and 300extractions. 0 C overnight and filtered. The ethylene di hlqr ee, w sthe; remo ed. under .v wum- .Th 'semi 'concent'rate thus produced 'wasthen examples. The following resultswere obtained: A

soluble in a polar solvent,- separatingthe soap from the u-nsaponifieclresidue and treating the unsaponified residue with a highly polarselective solvent which is characterized by being miscible withfat-soluble vitamin alcohols but immiscible with fat soluble vitaminesters to remove'that portion of the residue soluble therein and producea concentrate high in vitamin ester content.

2. A process for the production of fat soluble vitamin. esterconcentrates from vitamin containing material of marine origincomprising partiallyrsaponifying said material while cooling the same tobelow approximately 35 C.- so as to ren= der a relatively largeproportion of the material other than the vitamin ester and soap formedsoluble in a. polar'solvent, separating the soap from the unsaponifiedresidue and treating. the unsaponified residue with a highly polarselective solvent which is characterizedv by being miscible with fatsoluble vitamin alcohols, but immiscible with fat soluble vitamin estersto remove that portion of the residue soluble therein and produceaconcentrate high'in vitamin ester content.

3. A process for the production of fat soluble vitamin esterconcentrates from vitamin containing material.. ofmarine origincomprising par: tially saponifying while cooling the material so as torender a, relatively large proportion of the material other thanthe'vitamin ester and soap formed soluble in methanol, separating thesoap from the unsaponified residuevand treating the unsaponified residuewith methanol to remove that portion of the residue soluble therein and.produce a. concentrate high invitamin ester concc. forsubsequent Thecombined extracts were kept at tracted witlrmethanol as in the precedingex 'Units-of vitamin Apergram- Vitamin potency of semi-concentratel252,000 Vitamin potency of alcohol (methanol extract) 1 192,000 Vitaminpotency -of' ester-(methanolresidue) 320,000

Overallyield of vitamin" A was approximately As 'will be noted from theabove examples, in each case the potency of theesterconcentrate producedwas higher than that of the semi-'concentrate. In other words, even'withthe removal of the vitamin A alcohol suflicient'methan'ol'solublematerial was removed so that the the residue was enhanced. As previouslypointed potency of out in-the present specification, it is believed thatthis 'solubilizationeflect on the nonsaponified' and nonvitamin materialin the semi-concentrate is probably due to the formation of mono-anddiglycerides during the low temperature saponification process of thepresent invention. How-t ever it is'not desired to be limited by-thistheory.

.Having described our inventio' what we claim as new and desire tobe'secured by Letters Patent v '1 A process forthe production of fat,soluble vitamin ester concentrates from vitamin containingQmateriaI ofmarine ori'tin comprising partially sapon i inc w ecoo ns t e m e a s ast same'to below approximately 35C. so as to jre n"-.

der a relatively large proportion of the material otherzthan the vitaminester and soap formed soluble'in methanol, separating the soap from'theunsaponifiecl residue and treating the unsaponi: fled residue withmethanol to remove that portion of the residue soluble thereinandproduce a concentratehigh in'vitamin ester content.

Aprocess ,for the production of fat soluble vitamin ester concentratesfrom vitamin containing material of marine origin comprising partially'saponiiying while cohli'ng the material so as to render a relativelylarge proportion of the material other than the vitamin ester and soapformed soluble in ethanol; separating the soap from the 'unsaponifiedresidue and treating the tially saponifying said material. while coolingthe same to belowapproximately- 35". Geo as to render a relatively largeproportion of the material other than the vitamin ester and soap formedsol-- *uble in ethanol, separating the soap from themsaponified residueand treating the 'unsaponified. residuewith ethanolto remove thatportion of centrate-high' in vitamin ester content. t 7. A process forthe. production of fat solublev tamin ester concentrates f rom fishliver oils the residue soluble therein and .produce a contherein andprowhich comprises partially saponifying a fish liver oil while coolingthe same to maintain the temperature substantially constant duringsaponification so as to render a relatively large proportion of thematerial other than the vitamin ester and soap formed soluble in a polarsolvent, separating the soap from the unsaponified residue and treatingthe unsaponified residue with a highly polar selective solvent which ischaracterized by being miscible with fat-soluble vitamin alcohols butimmiscible with fat soluble vitamin esters to remove that portion of theresidue soluble therein and produce a concentrate high in vitamin estercontent.

8. A process for the production of fat solublevitamin ester concentratesfrom vitamin containin material of marine origin comprising parthe soapfrom the unsaponifled residue and treating the unsaponified residue witha highly polar selective solvent which is characterized by beingmiscible with fat-soluble vitamin alcohols but immiscible with fatsoluble vitamin esters to remove that portion of the residue solubletherein and produce a concentrate high in vitamin ester content. I

'9. A process for the production of fat soluble vitamin esterconcentrates from vitamin containing material of marine origincomprising par tially saponifying the said material while maintainingthe temperature thereof during saponification between approximately 0-25C., separating the. soap from the unsaponifled residue and treating theunsaponified residue with methanol to remove that portion of the residuesoluble therein and produce a concentrate high in vitamin ester content.

10. A process 101' the production of fat soluble vitamin esterconcentrates from vitamin containing materialof marine origin comprisingpartially saponifying the said material while maintaining thetemperature thereof during saponification between approximately 0-25 C.,separating the soap from the unsaponified residue and treating theunsaponifled residue with ethanol to. remove that portion of the residuesoluble therein and produce a concentrate high in vitamin ester content.

CHARLES E. DRYDEN. LORAN O. BUX'I 'ON.

